Background and importance Telephone calls are often patients' first healthcare service contact, outcomes associated with waiting times are unknown. Objectives Examine the association between waiting time to answer for a medical helpline and 1- and 30-day mortality. Design, setting and participants Registry-based cohort study using phone calls data (January 2014 to December 2018) to the Capital Region of Denmark's medical helpline. The service refers to hospital assessment/treatment, dispatches ambulances, or suggests self-care guidance. Exposure Waiting time was grouped into the following time intervals in accordance with political service targets for waiting time in the Capital Region: <30 s, 0:30-2:59, 3-9:59, and ≥10 min. Outcome measures and analysis The association between time intervals and 1- and 30-day mortality per call was calculated using logistic regression with strata defined by age and sex. Main results In total, 1 244 252 callers were included, phoning 3 956 243 times, and 78% of calls waited <10 min. Among callers, 30-day mortality was 1% (16 560 deaths). For calls by females aged 85-110 30-day mortality increased with longer waiting time, particularly within the first minute: 9.6% for waiting time <30 s, 10.8% between 30 s and 1 minute and 9.1% between 1 and 2 minutes. For calls by males aged 85-110 30-day mortality was 11.1%, 12.9% and 11.1%, respectively. Additionally, among calls with a Charlson score of 2 or higher, longer waiting times were likewise associated with increased mortality. For calls by females aged 85-110 30-day mortality was 11.6% for waiting time <30 s, 12.9% between 30 s and 1 minute and 11.2% between 1 and 2 minutes. For calls by males aged 85-110 30-day mortality was 12.7%, 14.1% and 12.6%, respectively. Fewer ambulances were dispatched with longer waiting times (4%/2%) with waiting times <30 s and >10 min. Conclusion Longer waiting times for telephone contact to a medical helpline were associated with increased 1- and 30-day mortality within the first minute, especially among elderly or more comorbid callers.